The in vitro fidelity of yeast DNA polymerase δ and polymerase ε holoenzymes during dinucleotide microsatellite DNA synthesis

Elucidating the sources of genetic variation within microsatellite alleles has important implications for understanding the etiology of human diseases. Mismatch repair is a well described pathway for the suppression of microsatellite instability. However, the cellular polymerases responsible for generating microsatellite errors have not been fully described. We address this gap in knowledge by measuring the fidelity of recombinant yeast polymerase δ (Pol δ) and ? (Pol ?) holoenzymes during synthesis of a [GT/CA] microsatellite. The in vitro HSV-tk forward assay was used to measure DNA polymerase errors generated during gap-filling of complementary GT(10) and CA(10)-containing substrates and ～90 nucleotides of HSV-tk coding sequence surrounding the microsatellites. The observed mutant frequencies within the microsatellites were 4 to 30-fold higher than the observed mutant frequencies within the coding sequence. More specifically, the rate of Pol δ and Pol ? misalignment-based insertion/deletion errors within the microsatellites was ～1000-fold higher than the rate of insertion/deletion errors within the HSV-tk gene. Although the most common microsatellite error was the deletion of a single repeat unit, ～ 20% of errors were deletions of two or more units for both polymerases. The differences in fidelity for wild type enzymes and their exonuclease-deficient derivatives were ～2-fold for unit-based microsatellite insertion/deletion errors. Interestingly, the exonucleases preferentially removed potentially stabilizing interruption errors within the microsatellites. Since Pol δ and Pol ? perform not only the bulk of DNA replication in eukaryotic cells but also are implicated in performing DNA synthesis associated with repair and recombination, these results indicate that microsatellite errors may be introduced into the genome during multiple DNA metabolic pathways.     

Beyond translesion synthesis: polymerase κ fidelity as a potential determinant of microsatellite stability

Microsatellite DNA synthesis represents a significant component of human genome replication that must occur faithfully. However, yeast replicative DNA polymerases do not possess high fidelity for microsatellite synthesis. We hypothesized that the structural features of Y-family polymerases that facilitate accurate translesion synthesis may promote accurate microsatellite synthesis. We compared human polymerases κ (Pol κ) and η (Pol η) fidelities to that of replicative human polymerase δ holoenzyme (Pol δ4), using the in vitro HSV-tk assay. Relative polymerase accuracy for insertion/deletion (indel) errors within 2-3 unit repeats internal to the HSV-tk gene concurred with the literature: Pol δ4 > Pol κ or Pol η. In contrast, relative polymerase accuracy for unit-based indel errors within [GT](10) and [TC](11) microsatellites was: Pol κ ≥ Pol δ4 > Pol η. The magnitude of difference was greatest between Pols κ and δ4 with the [GT] template. Biochemically, Pol κ displayed less synthesis termination within the [GT] allele than did Pol δ4. In dual polymerase reactions, Pol κ competed with either a stalled or moving Pol δ4, thereby reducing termination. Our results challenge the ideology that pol κ is error prone, and suggest that DNA polymerases with complementary biochemical properties can function cooperatively at repetitive sequences.     

The human gastric cancer-associated DNA polymerase β variant D160N is a mutator that induces cellular transformation

Approximately 30% of human tumors sequenced to date harbor mutations in the POLB gene that are not present in matched normal tissue. Many mutations give rise to enzymes that contain non-synonymous single amino acid substitutions, several of which have been found to have aberrant activity or fidelity and transform cells when expressed. The DNA Polymerase β (Pol β) variant Asp160Asn (D160N) was first identified in a gastric tumor. Expression of D160N in cells induces cellular transformation as measured by hyperproliferation, focus formation, anchorage-independent growth and invasion. Here, we show that D160N is an active mutator polymerase that induces complex mutations. Our data support the interpretation that complex mutagenesis is the underlying mechanism of the observed cellular phenotypes, all of which are linked to tumorigenesis or tumor progression.     

DNA polymerase kappa produces interrupted mutations and displays polar pausing within mononucleotide microsatellite sequences

Microsatellites are ubiquitously present in eukaryotic genomes and are implicated as positive factors in evolution. At the nucleotide level, microsatellites undergo slippage events that alter allele length and base changes that interrupt the repetitive tract. We examined DNA polymerase errors within a [T]₁₁ microsatellite using an in vitro assay that preferentially detects mutations other than unit changes. We observed that human DNA polymerase kappa (Pol κ) inserts dGMP and dCMP within the [T]₁₁ mononucleotide repeat, producing an interrupted 12-bp allele. Polymerase β produced such interruptions at a lower frequency. These data demonstrate that DNA polymerases are capable of directly producing base interruptions within microsatellites. At the molecular level, expanded microsatellites have been implicated in DNA replication fork stalling. Using an in vitro primer extension assay, we observed sequence-specific synthesis termination by DNA polymerases within mononucleotides. Quantitatively, intense, polar pausing was observed for both pol κ and polymerase α-primase within a [T]₁₁ allele. A mechanism is proposed in which pausing results from DNA bending within the duplex stem of the nascent DNA. Our data support the concept of a microsatellite life-cycle, and are consistent with the models in which DNA sequence or secondary structures contributes to non-uniform rates of replication fork progression.     

Clark's Nutcrackers Nucifraga columbiana Remember the Size of Their Cached Seeds

Clark's nutcrackers ( Nucifraga columbiana ) hide thousands of seeds in subterranean caches that they later recover using spatial information about cache location. In two experiments, we tested whether nutcrackers also remember another type of information regarding their caches – the size of the seeds in each cache. We videotaped birds during cache recovery and then measured their bill gape during probing behaviour as captured on the videotape. In experiment one, six birds each experienced two treatments: one that allowed them to cache and then recover large seeds, and the other, an identical treatment using small seeds. During this experiment, all six birds used a wider gape when attempting to recover seeds during the large-seed treatment than during the small-seed treatment, and gape width was significantly correlated with seed size. During experiment two, we presented birds with both large and small seeds within the same caching session. We also increased the retention interval between caching and recovery. These modifications increased the difficulty of the task. Six of the seven birds used a wider gape during seed recovery when digging for caches that contained large seeds than they did when searching for small seeds. The ability to remember the size of seeds placed within caches may serve to increase the likelihood of speedy and successful recovery. It also allows the birds another level of organization of their food supply. These are the first experiments to suggest that Clark's nutcrackers remember more about their caches than location alone.     

Call Convergence within Groups of Female Budgerigars (Melopsittacus undulatus)

The budgerigar (Melopsittacus undulatus) is a promising model species for the study of adult vocal learning. To date, several studies have confirmed the existence of vocal plasticity and, more importantly, rapid imitation of contact calls by adult male budgerigars. Vocal learning has not been investigated in female budgerigars, however. Since one likely function of the contact call is to denote group affiliation, we tested the hypothesis that female budgerigars, when placed into groups, would develop a shared contact call. We recorded the contact call repertoires of eight adult female budgerigars that were unfamiliar with one another, then placed them into two groups. Each group was deprived of visual contact with other birds. Recording sessions continued for the subsequent 8 wks, and behavioral observations were also conducted during this time. Within 4–7 wks, females in both groups converged on a common call type. This rate of convergence is slower than that observed in prior experiments limited to male birds, and much slower than vocal imitation by male budgerigars paired with females. Therefore, while our study documents vocal plasticity in adult female budgerigars, it also suggests that female budgerigars learn new vocalizations more slowly than males do.     

CO2 supplementation eliminates sugar-rich media requirement for plant propagation using a simple inexpensive temporary immersion photobioreactor

Plant Cell, Tissue and Organ Culture (PCTOC) - In vitro plant propagation systems such as temporary immersion bioreactors (TIBs) are valuable tools that enable production of disease-free plants...     

Misalignment-mediated DNA polymerase beta mutations: comparison of microsatellite and frame-shift error rates using a forward mutation assay

Mutations arising in microsatellite DNA are associated with neurological diseases and cancer. To elucidate the molecular basis of microsatellite mutation, we have determined the in vitro polymerase error frequencies at microsatellite sequences representative of those found in the human genome: [GT/CA](10), [TC/AG](11), and [TTCC/AAGG](9). DNA templates contained the microsatellites inserted in-frame into the 5' region of the herpes simplex virus thymidine kinase (HSV-tk) gene. Polymerase beta (polbeta) error frequencies were quantitated in microsatellite sequences, relative to frame-shift error frequencies in coding sequences, from the same DNA synthesis reaction. The polbeta error frequencies within the dinucleotide sequences were (2-9) x 10(-3), 14-72-fold higher than the ssDNA template frequencies. The polbeta error frequencies within the tetranucleotide sequences were (4-6) x 10(-3), a 4-13-fold increase over background. Strand biases were observed for the [TC/AG](11) and [TTCC/AAGG](9) alleles, in which more errors were produced when the purine strand served as a template. Mutations within each microsatellite included noncanonical base substitution events and single nucleotide deletions as well as the expected unit length changes. An exponential relationship was observed between the polymerase error frequency per site and both the number of repetitive units and total length of the allele. Our observations are consistent with the strand slippage model of microsatellite mutagenesis and demonstrate that DNA sequence and/or structural differences result in mutational strand biases. To our knowledge, this is the first direct quantitation of DNA polymerase errors in vitro using template microsatellite sequences.     

FLAGELLAR APPARATUS STRUCTURE IS SIMILAR BUT NOT IDENTICAL IN VOLVULINA STEINII,EUDORINA ELEGANS,AND PLEODORINA ILLINOISENSIS (CHLOROPHYTA): IMPLICATIONS FOR THE “VOLVOCINE EVOLUTIONARY LINEAGE”1

The colonial and multicellular members of the Volvocales can be arranged in order of increasing size and complexity as the “volvocine series.” This series is often assumed to reflect an evolutionary progression. The flagellar apparatuses of previously examined algae are not consistent with a simple lineage. The flagellar apparatuses of Astrephomene gubernaculifera Pocock, Gonium pectorale Müller, Platydorina caudata Kofoid, Volvox rousseletii G. S. West, and V. carteri f. weismannia (Powers) Iyengar differ from one another, and there is no apparent progression inflagellar apparatus features from the simple to complex colonial forms. We examined the flagellar apparatuses of Volvulina steinii Playfair, Eudorina elegans Ehr., and Pleodorina illinoisensis Kofoid and found them to be similar to one another. The basal bodies are connected by a distal fiber that is offset to the anti side of the cell. Two microtubular rootlets originate on the inside of the basal bodies and extend toward the syn side. The other two rootlets are oriented perpendicular to the first two and are anti-parallel to each other. A coarsely striated component underlies the four-membered rootlets and extends to the basal bodies. A proximal fiber complex connects the two basal bodies. This complex consists of a branched striated component on the cis side of each basal body. One part extends toward the anti side of the cell, while the other extends into a fibrous component that runs between basal bodies. An additional structure extends in the anti direction from the trans side of each basal body. A fibrous component extends past one basal body in all four species. This component goes past the trans basal body in Volvulina steinii and the cis basal body in E. elegans and P. illinoisensis. The flagellar apparatuses of these organisms are similar to those of G. pectorale and Volvox carteri but different from the other colonial volvocalean algae examined. The algae examined in this study plus G. pectorale and V. carteri probably share a common evolutionary history that postdates the transition from the unicellular to colonial habit. Such a shared evolutionary history is a requirement of the volvocine hypothesis. However, we have not observed progressive changes in the flagellar apparatus correlated with increasing cell number, differentiation, and sexual specialization. Thus, it is possible, but not certain, that G. pectorale, Volvulina steinii, E. elegans, P. illinoisensis, and Volvox carteri may form part of a volvocine lineage.     

Positive correlation between DNA polymerase alpha-primase pausing and mutagenesis within polypyrimidine/polypurine microsatellite sequences

Microsatellite DNA sequences are ubiquitous in the human genome, and mutation rates of these repetitive sequences vary with respect to DNA sequence as well as length. We have analyzed polymerase-DNA interactions as a function of microsatellite sequence, using polypyrimidine/polypurine di- and tetranucleotide alleles representative of those found in the human genome. Using an in vitro primer extension assay and the mammalian DNA polymerase alpha-primase complex, we have observed a polymerase termination profile for each microsatellite that is unique to that allele. Interestingly, a periodic termination profile with an interval size (9-11 nucleotides) unrelated to microsatellite unit length was observed for the [TC](20) and [TTCC](9) templates. In contrast, a unit-punctuated polymerase termination profile was found for the longer polypurine templates. We detected strong polymerase pauses within the [TC](20) allele at low reaction pH which were eliminated by the addition of deaza-dGTP, consistent with these specific pauses being a consequence of triplex DNA formation during DNA synthesis. Quantitatively, a strand bias was observed in the primer extension assay, in that polymerase synthesis termination is more intense when the polypurine sequence serves as the template, relative to its complementary polypyrimidine sequence. The HSV-tk forward mutation assay was utilized to determine the corresponding polymerase alpha-primase error frequencies and specificities at the microsatellite alleles. A higher microsatellite polymerase error frequency (50x10(-4) to 60x10(-4)) was measured when polypurine sequences serve as templates for DNA synthesis, relative to the polypyrimidine template (18x10(-4)). Thus, a positive correlation exists between polymerase alpha-primase pausing and mutagenesis within microsatellite DNA alleles.